This invention relates to plant tissue culture conditions designed to more efficiently obtain transgenic plant cells, and more particularly to the use or incorporation of a low-oxygen environment in which to culture plant cells before transformation or during selection and regeneration.
The ability to transfer genes from a wide range of organisms to crop plants by recombinant DNA technology has become widespread in recent years. This advance has provided enormous opportunities to improve plant resistance to pests, disease, and herbicides, and to modify biosynthetic processes to change the quality of plant products. A highly efficient method for transformation of these crop plants continues to be a goal as there is a need for high capacity production of economically important plants.
Several technologies for the introduction of DNA into cells are well known to those of skill in the art and can be divided into categories including but not limited to: (1) chemical methods; (2) physical methods such as microinjection, electroporation, and particle bombardment; (3) viral vectors; (4) receptor-mediated mechanisms; and (5) Agrobacterium-mediated plant transformation methods.
Agrobacterium-mediated transformation is the most widely used method for transforming crop plants. Several Agrobacterium species mediate the transfer of a specific DNA known as “T-DNA”, that can be genetically engineered to carry a desired piece of DNA into the selected plant species. The major events marking the process of T-DNA mediated pathogenesis and ultimate transformation are induction of virulence genes, processing and transfer of the T-DNA to the plant's genome.
Typically, Agrobacterium-mediated genetic transformation of plants involves several steps. The first step, in which the Agrobacterium and plant cells are first brought into contact with each other, is generally called “inoculation.” Sometimes, the plant cells are “precultured” before inoculation. This is often to condition the cells to be more amenable to transformation. Following the inoculation step, the Agrobacterium and plant cells/tissues are usually grown together for a period of several hours to several days or more under conditions suitable for growth and T-DNA transfer. This step is termed “co-culture”. Following co-culture and T-DNA delivery, the plant cells are often treated with bactericidal or bacteriostatic agents to prevent further growth of the Agrobacterium. If this is done in the absence of any selective agents to promote preferential growth of transgenic versus non-transgenic plant cells, then this is typically referred to as the “delay” step. If done in the presence of selective pressure favoring transgenic plant cells, then it is referred to as a “selection” step. When a “delay” is used, one or more “selection” steps usually follow it. Both the “delay” and “selection” steps typically include bactericidal or bacteriostatic agents to prevent further growth of any remaining Agrobacterium cells because the growth of Agrobacterium cells is undesirable after the infection (inoculation and co-culture) process. Then the selected transgenic cells are put through a “regeneration” step in which transformed plantlets are produced.
Particle bombardment is another common method of transforming plants. In this method, particles are coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, or preferably, gold. An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System (BioRad, Hercules, Calif.), which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with plant cells cultured in suspension. Microprojectile bombardment techniques are widely applicable and may be used to transform virtually any plant species. After bombardment of the plant tissues or cells, selection and regeneration are performed as described above for Agrobacterium transformation.
The present invention provides a novel improvement to the transformation process by providing a low-oxygen environment to the plants cells or tissue during at least one of the preculture, selection or regeneration phases of transformation. A continuing need exists in the field of plant transformation to produce a better quality of cell culture and to maximize genotypes with poor culture response or poor regeneration capabilities.